The inward rectifier current (IK1) plays a role in the excitability properties of the cardiac cell. Loss of function in one of the genes (KCNJ2) underlying the channel protein (Kir2.1) in humans leads to Andersen's syndrome, characterized by dysmorphic features, paralysis and cardiac arrhythmias. Properties of ion channels depend on their three dimensional structure as well as by their interactions with accessory proteins. There is little information available on proteins that directly associate with Kir2.x channels, or how these proteins modify channel function in the heart. The scaffolding proteins, (SAP97, Veli and CASK) that target, cluster and regulate ion channels, have been shown to directly bind or are co localized with Kir2.x channels in the heart, with implications that are largely unknown. Our objective is to determine the protein-protein interactions involved in the regulation of Kir2.x channel function. Our main hypothesis is that SAP97, Veli and CASK interactions with Kir2.x channels results in modification of channel biophysics, regulation, expression and subcellular targeting in the heart. We will use a combination of approaches including electrophysiological, surface plasmon resonance, biochemical and molecular biological techniques and carry out our studies in HEK293 cells and in isolated cardiac myocytes. Specific Aims: 1) To investigate the molecular basis of changes in the biophysical properties of Kir2.x channels following co-expression with cardiac PDZ domain proteins. 2) To study the impact of interactions between Kir2.x channels and PDZ domain proteins on channel phosphorylation by PKA. 3) To determine the role of PDZ domain proteins on Kir2.x channel expression, localization and function in cardiac and non-cardiac cells. These studies should lead to a better understanding of the molecular properties that modulate the function of the classical cardiac inward rectifier current.